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Questions related from Enes Yağız Akdaş
Dear all, I cloned 25bp fragment into a vector. My no-insert ligation has no colonies and my insert+vector ligation has many. Which is the first clue that I have cloned the fragment. Than I...
12 December 2019 7,634 8 View
Dear friends, We kindly get U-251 MG cell line as a gift from an other lab. after 3 passage we have determined that cell line has yeast contamination. Each stock is contaminated while other...
12 December 2018 5,404 8 View
Dear all, I have generated Stable Cell lines via G418 selection. I have used pCMV-miR vector.Except Neo-GFP region, my chance is 2/5 to integrate genome without damaging CMVpromotor-insert-PolyA...
08 August 2018 8,155 2 View
hey, I have generated stable cell lines with random entegration of pCMV-miR vector. Vector contains a neoR gene fused with GFP with IRES. I got some GFP+ and GFP- colonies. and GFP- colonies...
08 August 2018 663 3 View
Dear Friends, I performed a kill-curve experiment and determinated that ; at 400mg/ml g418 consantration only 5-10 non-profilable cell survived at 7 day in 48 well-plate, plated with 50.000 U87...
06 June 2018 8,063 3 View
Hey, I have a small problem. I have cloned my target gene's UTR into pmir-Glo vector. But the problem is that frefly luciferase gene also contains miRNA target sequence. Thus when I clone my UTR...
05 May 2018 9,147 0 View
Hey friends, I am working with U-87 cell line. Standart transfection reagents show no transfection efficiency on U-87 cells. We have Amaxa nucleofector but its reagents are highly...
04 April 2018 9,701 0 View
Dear all, I am working with U-87 MG (a glioblastoma cell line whose origine is unknown but male) and T-98G. I have clonned my miRNA genes into pCMV-miR vector that express transgene via CMV...
04 April 2018 6,786 3 View
I am working with glioblastoma U-87 cell lines. I have pCMV-miR expression vector ~8kb. I tired both Transfast, Fugene HD, PEI, Jetprime. They had max %30 efficiency. Transfast best with 1:1...
04 April 2018 9,687 8 View
Hey all, To overexpress a miRNA; instead of pre-miRNA sequences, pri-miRNA sequences are also being used driven by RNAP II promoters. in articles these fragments nearly miRNA+100-300 bp flanking...
04 April 2018 3,834 5 View
Hey, I am trying to clone a 600 bp fragment into pcmv-mir vector. I use 30mg/L kanamycin in my agar plates. I digest 1 uq plasmid with 10 unit of enzyme (Sgf I and MluI) over night. I run a...
03 March 2018 4,832 7 View
Hey, I have a problem during my plasmid electrophoresis. I got my plasmid band in the same line with 15kb marker. But when I digest it with a restriction enzyme I got my band in the correct...
02 February 2018 6,189 7 View
Hey, I have a phagemid vector with ColE1 origin (pCMV-miR) normally my vector is 6.2kb and when I ligate it with a miRNA precurser it became 7kb. My problem is during electrophoresis, I got an...
02 February 2018 551 0 View
Dear people, I have a plasmid (pCMV-miR) that contains, f1 ori, ColE1 ori as well as SV40 ori. As I thought it, my plasmid is a cosmid (Phagemid) with very low copy number. When I transform my...
02 February 2018 7,610 1 View
I have a plasmid isolation that probably contains salt contamination. Due to the fact that , all my plasmid is digested because of Star activity at 50uL reaction overnight digestion. I digest 3ug...
01 January 2018 7,558 3 View
Hi everyone, I am digesting 1 ug of pmiR-glo vector with Xhoi and SacI restriction enzymes. When I started to minimize my volume (50 to 30) I started to get strange bands. Can this be a result...
12 December 2017 6,289 1 View
I will clone my miRNA targeted 3'UTRs into a lusiferase reporter vector ( miR-glo lusiferase reporter) Unfortunatelly, I dont have primers to check cloning via PCR. Due to the fact that , I...
11 November 2017 3,036 4 View
Hey, I have clonned a double stranded oligo into glo-mir vector. My vector is 7.5kb and insertion is 81bp. I have isolated my plasmid from colonies after trsnsformation and perform agarose gel...
11 November 2017 6,068 4 View
Does anyone know the exact chemicals in qiagen lyseblue or other kits "blue" chemical in resuspention buffers? I am trying to perform alkaline lysis. But unfortunatelly I couldn't find the right...
11 November 2017 1,670 2 View
Hey, I will clone a miRNA into the expression vector. I have 2 primer sets to get full lenght pre-miRNA, as it is shown belove; Set 1: 5' _____________3' Set 2: 3'...
10 October 2017 9,469 7 View
I wonder that , does the DNA polymerase deattach from the DNA double helix after the sequens between two primer is complated? What I am trying to ask is that, If I have a 200 nt single strand...
07 July 2017 1,236 4 View
I am working with a protein whose 3' UTR sequence is targeted by some of the miRNAs. I will perform luciferase reporter assay to be sure about that these miRNAs cut my mRNA's 3'UTR sequence. But...
07 July 2017 5,943 4 View
Dear friends; I have monoclonal CTL2 antibody. And it is known that CTL2 protein has only 2 isoforms thar are only ~5 aminoacid changed. Bu after 14h incubation at +4 with primary antibody, I get...
03 March 2017 7,515 9 View
I have an acid-phenol:chloroform but its pH isn't acidic anymore, can I change the pH with HCL?
02 February 2015 4,397 3 View
